1. Field of the Invention
The present invention relates to conformational protein isomers, to methods of producing and isolating such isomers, to methods of utilizing such isomers, and to products comprising and products made from such isomers. In another aspect, the present invention relates to conformational protein isomers having at least one disulfide bond, to methods of producing and isolating such disulfide isomers, to methods of utilizing such disuflide isomers, and to products comprising and products made from such disulfide isomers.
2. Description of the Related Art
A protein can potentially assume an exceedingly large number of conformations. Under physiological conditions, a protein usually folds “properly” and adopts the native structure with a well defined three dimensional conformation. Unlike the native protein, a denatured protein consists of a collection of conformational isomers that exist in a state of equilibrium. Conformational isomers of denatured proteins are rich in number and varied in shape. Conformational isomers represent an opulent resource of biological molecules that have, thus far, remained untapped. The major obstacle in utilizing the untapped potential of conformation isomers is the inherent difficulty in the isolation and characterization of pure conformational isomers, not only because of the excessive large number that may exist but also because of their instability and rapid inter-conversion.
One conventional approach used to study protein folding is to unfold proteins in the presence of a strong denaturant, such as 8M urea or 6. m GdmCl, by extreme pH, or by high temperature. Following the removal of the denaturant, reduction of pH, or reduction of temperature, the denatured proteins usually refold spontaneously to form the native structure. The refolding pathway of the protein is monitored by the restoration of at least one physicochemical signal that distinguish the native and unfolded states. Commonly used signals are spectra of fluorescence, circular dichroism, infrared, ultraviolet light and NMR coupled with amide proton exchange. Unfortunately, in most cases this method does not permit isolation of folding intermediates.
Another conventional method use to study protein folding is oxidative folding of disulfide containing proteins. Proteins are reduced and denatured in the presence of reducing agent, such as dithiothreitol, and denaturant, such as 6M GdmCl. After exclusion of the reductant and denaturant, the reduced and denatured protein is allowed to refold in the presence of redox buffer. The refolding pathway is then tracked by the mechanisms of formation of the native disulfide bonds. For example, a protein that contains three disulfide bonds can potentially assume 75. different disulfide isomers (15. isomer species having one disulfide bond, 45. having two disulfide bonds, and 15. having three disulfide bonds). The disulfide folding pathway is characterized by the heterogeneity and structures of the disulfide isomers that accumulate in the process of oxidative folding that leads to formation of the native structure. However, without chemical modification, the method of oxidative folding does not generate stable isomers.
In spite of advancements in the art, methods for generating large numbers of stable conformational isomers of a protein have not been developed. Thus, there remains a need for methods for producing large numbers of stable conformational isomers of a protein.
There is another need in the art for purified populations of stable conformational isomers of a protein.
There is even another need in the art for methods for screening and identifying therapeutic agents/drugs wherein the agent is a disulfide isomer.
There is still another need in the art methods for screening and identifying therapeutic agents/drugs wherein the agent is a protein stabilizer.
There is yet another need in the art for methods of investigating the molecular mechanisms of a conformational disease.
There is even still another need in the art for compositions and products comprising protein conformational isomers.
There is even yet another need in the art for methods for treating a patient afflicted with a conformation disease.
These and other needs will become apparent to those of skill in the art upon review of this specification, including its drawings and claims.